Zhiliang Zhang, 1,2 Yongqiang Wen, 1, a ) Ying Ma, 1 Jia Luo, 1 Xingye Zhang, 1 Lei Jiang, 1 Yanlin
Song 1, b)
1 Beijing National Laboratory for Molecular Sciences (BNLMS), Key Lab of Organic Solids, Laboratory of New Materials,
Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
2 Research Center of Analysis and Test, Shandong Institute of Light Industry, Jinan 250353, China.
Email: ylsong@iccas.ac.cn, wyq_wen@iccas.ac.cn
Tel/Fax: 86-10-6252 9284
1. Materials and methods
1.1. Materials
Silver nitrate, 2-nitro-4-(trifluoromethyl) thiophenol (NFMT), and sodium citrate tribasic dihydrate were purchased from Aldrich. 1,2-dithiane-4-O-dimethoxytrityl-5-
[(2-cyanoethyl)-N,N-diisopropyl]-phosphoramidite (DTPA) was synthesized according to the literature procedures (Liepold et al, 2008). The oligonucleotides used were purchased from TaKaRa Biotech (Dalian, China) and used as received. DNA oligonucleotides:
5'-TTCCCCCCTTTTT(DTPA)
3
(DTPA represent the cyclic disulfide-containing phosphate derivative); 5'-TTCCCCCCTTTTT(CH
2
)
6
SH; control sequence: 5'-TTCAGATATTTTT(DT
PA)
3
. Silver colloids with mean diameters of 30 nm were synthesized by the citrate method.
S1
All buffers were prepared with ultra-pure MilliQ water (resistance > 18 MΩ cm
-1
).
1.2. Apparatus
Transmission electron microscopy (TEM) images were obtained using a Philips CM 200 kV electron microscope. UV-Vis spectra were collected using a Hitachi U-4100 spectrophotometer. Raman spectra were taken on a Renishaw invia Raman Microscope using the 532 nm laser excitation with a 50 × objective. The Raman light was dispersed by a diffraction grating with 1800 lines/mm. Data acquisition time: 10 s. Circular dichroism (CD) spectra were measured using a JASCO J-815 circular dichroism spectrometer.
1

2. Experimental details and mechanism discussion
2.1 Preparation of labeled DNA-AgNPs conjugates
30 nm AgNPs (1 mL, 0.5 nM) was mixed with purified DNA strands (10 nmoles). The mixture was incubated in 50 mM phosphate buffer. Then the NaCl concentration was gradually increased to 0.3 M by adding 1 M NaCl over 48 h. The mixture was centrifuged at
13000 rpm for 20 min. The supernatant containing free oligonucleotides was removed. The precipitates containing DNA-AgNP conjugates were washed with phosphate buffer containing 0.3 M NaCl twice and finally redispersed in the same buffer. A freshly prepared reporter solution (3 mM in methanol) was added dropwise to a solution of DNA-AgNP conjugates. The mixture was gently stirred for 24 h. The solution was then centrifuged for 20 min at 13000 rpm, and the supernatant was removed.
2.2. Stability studies of functionalized AgNPs conjugates
The stability of the conjugated AgNPs was evaluated by assessing the formation of AgNP aggregates in 50 mM phosphate buffer at different salt concentration at 40 ºC. The stability of the nanoparticles was monitored by measuring the change in the plasmon band absorbance at 410 nm using UV-Vis spectrophotometer at different time.
Fig. S1.
UV-Vis extinction spectra changes of triple cyclic modified (a) and single thiol-modified ( b ) DNA conjugated AgNPs in 50 mM phosphate buffer at various salt concentrations: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 M. All the data were obtained after mixing the AgNPs in the solution for 48 hours.
2

2.3. Confirmation of the switching behavior of the DNA nanomachine
The pH-induced transformation of the DNA nanomachine used in our system was confirmed by CD spectroscopy. As shown in Fig. S2, when the system is in its initial state where the pH value is around 8.0, the CD spectrum shows positive and negative bands at 274 nm and 247 nm, respectively (curve I), indicating that the structure of DNA is random coil. When the pH of the solution was changed to slightly acid ic conditions
(pH 5.0), the CD spectrum shows a positive band at 285 nm, a crossover at around 271 nm, and a negative band centered at 256 nm (curve II), which is the characteristic spectrum of i-motif structures as reported previously.
S3
Fig. S2.
CD spectra of i-motif DNA measured in 10 mM phosphate buffer containing 0.3 M
NaCl at pH 8.0 (curve I) and the random coil conformation at pH 5.0 (curve II). DNA sequence: 5'-TTCCCCCCTTTTT(DTPA)
3
The pH-induced controlled aggregation/disosation of dye-coded AgNPs
by the adjustment of pH of aqueous media
was confirmed by UV-Vis spectra and TEM images
(Fig. S3). At the initial stage pH (pH 8.0), the UV-Vis spectrum displays a plasmon resonance at 410 nm (Fig. S3, curve I), indicating a nonaggregated state. When the pH was adjusted to 5.0, the AgNPs plasmon resonance broadened and redshifted (Fig. S3, curve II), indicating AgNPs were brought much closer to each other due to the formation of interparticle i-motif structure. The disperse and aggregated states of AgNPs can be clearly observed by the corresponding TEM images (Fig. S3).
3

Fig. S3.
Typical UV-Vis extinction spectra of Raman dye and DNA-functionalized AgNPs solutions at pH 8.0 (curve I) and at pH 5.0 (curve II), where they hybridized to form an i-motif conformation. The inset shows the typical TEM image of AgNPs at pH 8.0 and at pH
5.0.
2.4
Control experiments
Fig. S4.
CD spectra of non-i-motif DNA measured in 10 mM phosphate buffer containing
0.3 M NaCl at pH 8.0 (curve I) and at pH 5.0 (curve II). DNA sequence: 5'-
TTCAGATATTTTT(DTPA)
3
-3'
To demonstrate that the observed AgNP assembly is specific to the i-motif DNA, control experiments were performed.
The experimenal results indicate that the CD spectra of non-i-motif modified DNA do not undergo obvious change (Fig. S4) and the AgNPs functionalized with this kind of oligonucleotide do not aggregate when the pH is adjusted
4

from 8.0 to 5.0. No significant change in extinction spectra and SERS spectra was observed
(Fig. S5, Fig. S6 a)). This not only confirms that the color change and spectral shift observed for the i-motif DNA-modified AgNPs is specific to the i-motif DNAs, but also indicates that the AgNPs functionalized with triple cyclic disulfide-modified DNA are quite stable in the experimental conditions. Moreover, no obviously influence on the SERS effects was observed when the pH is adjusted from 8.0 to 5.0 for the nanoparticles modified with only
Raman reporter molecules (Fig. S6 b)).
Fig. S5.
UV-Vis extinction spectra of non-i-motif DNA measured in 10 mM phosphate buffer containing 0.3 M NaCl at pH 8.0 (curve I) and at pH 5.0 (curve II). DNA sequence:
5'-TTCAGATATTTTT(DTPA)
3
-3'
Fig. S6.
(a) SERS spectra of non-i-motif DNA and Raman dye-functionalized AgNPs obtained at pH 8.0 (black curve), and pH 5.0 (red curve), respectively. DNA sequence:
5'-TTCAGATATTTTT(DTPA)
3
-3'. (b) SERS spectra of Raman dye-functionalized AgNPs obtained at pH 8.0 (black curve), and pH 5.0 (red curve), respectively.
5

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